The invention relates to a Mycobacterium strain with modified erp gene and the vaccine composition containing same.
Tuberculosis is an infectious disease caused in most cases by inhalation of bacteria belonging to the complex of Mycobacterium tuberculosis species (M. africanum, M. bovis, M. tuberculosis). With eight million new human cases annually causing three million deaths worldwide, tuberculosis remains a major public health problem (Sudre et al., 1992). The discovery of effective antibiotics (streptomycin, isoniazide, rifampicin and the like) appeared to allow the eradication of this disease. However, it is estimated that currently only 50% of patients are diagnosed and receive treatment. This treatment is often inappropriate or poorly monitored and leads to the appearance of an increasing number of antibiotic-resistant and even polychemoresistant strains (Dooley et al., 1992). In this context, the development of a vaccinal prophylaxis appears as a preferred solution for the control and eradication of tuberculosis.
The fact that an attenuated pathogenic bacterium is used as a component of a vaccine has been widely described and implemented in the prior art. The methods for obtaining such attenuated bacteria comprise the random selection of mutants induced chemically or by irradiation, or the production of recombinant bacteria of pathogenic origin in which a gene involved in a metabolic pathway has been inactivated by genetic engineering.
Straley et al. (1984) have studied the survival of avirulent mutants of Yersinia pestis which are deficient in one or more metabolic pathways.
Noriega et al. (1994) have manufactured, by genetic engineering, an oral Shigella strain intended to be used as a vaccine prototype by introducing deletions into a gene (aroA) encoding a protein involved in a metabolic pathway for an aromatic amino acid and they have demonstrated that the resulting defective recombinant Shigella strains were capable of inducing protective antibodies against the wild-type pathogen.
A major study has also been carried out using Salmonella as a model. See for example the reports by Hoiseth et al. (1981), Levine et al. (1987), Oyston et al. (1995) and Curtiss (1990).
However, similar studies have not yet been carried out for Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), which infects a third of the world population and kills three million people per year. Tuberculosis is the most important cause of mortality in the world caused by a group of infectious organisms (Bloom and Murray, 1992) grouped under the name xe2x80x9cM. tuberculosis complexxe2x80x9d. According to the WHO, more people died of tuberculosis in 1995 than during any previous year. It has been estimated that up to half a billion people will suffer from tuberculosis in the next 50 years. However, in spite of its importance, the genetic determinants of the virulence and persistance of M. tuberculosis remain scarcely characterized.
Indeed, the virulence of pathogenic mycobacteria is associated with their ability to grow and persist at the intracellular level. Bacteria of the M. tuberculosis complex parasitize the phagocytic cells in which they live and multiply in a specialized vacuolar compartment called the phagosome. The phagosomes containing live M. tuberculosis do not acidify and escape fusion with the lysosomes. The mechanisms by which M. tuberculosis make their phagosome more hospitable remain unknown and the mycobacterial genes affecting their intracellular growth and multiplication are being actively investigated.
The extreme difficulty of creating defined mutants of M. tuberculosis, either by allelic exchange or by transposon mutagenesis, has prevented the identification of these virulence factors according to the postulates of Koch (Falkow, 1988; Jacobs, 1992). Alternative genetic strategies have been used instead, including the complementation of a non-pathogenic bacterium (Arruda et al., 1993) and of spontaneous avirulent mutants with virulent M. tuberculosis (Pascopella et al., 1994) and virulent M. bovis (Collins et al., 1995) chromosomal DNA libraries. Although these studies have identified genes potentially involved in the entry into the epithelial cells and conferring a growth advantage in vivo, the great majority of the mycobacterial genes involved in the virulence and survival in the host organism remain unknown. The development of effective mutagene systems is therefore the priority for mycobacterial genetics.
One method for the creation of mutants is allelic exchange mutagenesis. Recently, allelic exchanges taking place with a low frequency have been demonstrated in bacteria of the M. tuberculosis complex using a suicide vector (Reyrat et al., 1995; Azad et al., 1996) and novel protocols allowing easier detection of the allelic exchange mutants have also been developed (Norman et al., 1995; Balasubramamian et al., 1996; Pelicic et al., FEMS Microbiol. Lett. 1996). However, the detection of a very rare allelic exchange event is prevented by low transformation efficiencies and the high frequency of illegitimate recombinations. Thus, many Mycobacterium genes still remain refractory to allelic exchange by means of the available technologies.
More particularly, the allelic exchange mutagenesis systems require the use of more efficient methods. The problems encountered may be overcome by the use of a replicative vector which is effectively conditionally lost. The possibility of introducing such vectors makes it possible to avoid the problems resulting from the low transformation efficiencies. Thus, under counterselection conditions, the clones still containing the vector are eliminated, thus allowing the detection of very rare genetic events. Such a system has been recently developed. Using a replicative vector under certain conditions which is lost at 39xc2x0 C. in M. smegmatis, the first library of mycobacterial insertion mutants was constructed in this rapidly growing model strain (Guilhot et al., 1994). However, the heat-sensitive vectors used are only slightly heat-sensitive in slow-growing mycobacteria of the M. tuberculosis complex and therefore cannot be used in these species for allelic exchange mutagenesis (unpublished data).
The inventors have succeeded in altering the virulence and the persistance of Mycobacterium strains in the host cells.
They have indeed produced a Mycobacterium strain one gene of which has been modified so as to attenuate its virulence.
Modified gene is understood to mean a gene which has undergone a modification abolishing the production of the corresponding protein or allowing the production of a protein which is at least 20% different in terms of activity compared with the natural protein.
BCG (Bacille Calmette-Guxc3xa9rin), an avirulent strain derived from M. bovis, is widely used worldwide as a vaccine against tuberculosis. However, while BCG can be administered without any problem to individuals with no immune deficiency, the same is not true for immunosuppressed individuals such as people infected with the AIDS virus, people who have had a marrow transplant, people suffering from a cancer, or people with altered functioning of one of the components of the immune system.
That is the reason why the present invention relates to a Mycobacterium strain with limited persistence.
The gene modified in the Mycobacterium strain in accordance with the invention is the erp gene. It may also be a gene having a complementation homology (of at least 80%) with the erp gene.
Analysis of the deduced protein sequence of the erp gene shows that the latter encodes a protein whose calculated molecular mass is 28 kDa. The presence of a signal sequence for export at an N-terminal position as well as the existence of a C-terminal hydrophobic region suggest that the molecule can be anchored in the plasma membrane or located at the surface of the bacilli. Furthermore, the central region of the protein comprises two repeat regions each composed of 6 copies of a P(G/A)LTS (SEQ ID NO: 1) motif positioned in tandem. This organization is similar to that found in many surface proteins associated with peptidoglycan in Gram-positive bacteria and in Plasmodium.
A genetic methodology allowing the selection and the identification of DNA fragments encoding exported proteins has recently been adapted for M. tuberculosis in the laboratory. This system is based on the production of libraries of M. tuberculosis DNA fragments fused with the E. coli alkaline phosphatase (phoA) gene lacking expression and export signals. Alkaline phosphatase (PhoA) possesses detectable enzymatic activity only after export across the plasma membrane. Using this system, several DNA fragments allowing the export of PhoA in mycobacteria have been selected in the presence of a chromogenic substrate, and partially sequenced. One of the fusions carried by the recombinant plasmid pExp53 exhibits sequence similarities with an M. leprae gene which encodes a protein of 28 kDa, potentially located at the surface of the bacillus. Furthermore, this protein is a major M. leprae antigen recognized by the sera of lepromatous leprosy patients (WO 9607745). We have furthermore determined, by molecular hybridization experiments, that the erp gene is unique in the M. tuberculosis genome and that it is also present in the genome of the other members of this complex of species (M. africanum, M. bovis, M. bovis BCG).
To allow the study of ERP and to confirm its localization at the surface, specific anti-ERP antibodies were produced. For that, the ERP protein fused with the maltose-binding protein (MalE/MBP) or fused with a C-terminal peptide containing 6 histidine residues (SEQ ID NO: 2) was produced. This strategy made it possible to obtain, in a large quantity, recombinant ERP-MalE and ERP(His)6 proteins expressed in Escherichia coli. The purification of these molecules was carried out using the techniques of affinity chromatography on a resin of amylose (MalE system) or of chelated nickel (Histidine system). The relative molecular weight, determined by SDS-PAGE electrophoresis, is 36 K. The difference with the theoretical molecular weight may be attributed to a delay in electrophoretic migration due to the high content (15%) of proline residues. A protocol for immunizing rabbits with the aid of the purified ERP-MalE and ERP(His)6 chimeras made it possible to obtain polyclonal sera at a high titer which allow the specific detection of the ERP protein.
With the aid of the antisera obtained in rabbits, the localization of the ERP protein in Mycobacterium tuberculosis was specified. Electron microscopy observations after immunolabeling with colloidal gold made it possible to detect the presence of the ERP protein at the surface of tubercle bacilli derived from an in vitro culture. Thus, the ERP protein is capable of exhibiting at the surface of the mycobacteria epitopes of other antigens and for vaccinal or therapeutic purposes. Furthermore, similar experiments have made it possible to detect the ERP protein in murine macrophages infected with M. tuberculosis. 
To analyze the function of the ERP protein, a BCG strain in which the erp gene was modified by allelic exchange was constructed. The survival of this strain in comparison with the wild-type strain was analyzed in the mouse model. It was demonstrated that the mutation of the erp gene severely affects the persistance of M. bovis BCG. This reduction in persistance is observed in all the organs tested (spleen, liver, lungs). In addition to the role of the gene in the BCG survival process, these observations mean that the erp gene is expressed during the growth phase in vivo in the host.
More particularly, the modification of the erp gene is carried out by mutation, insertion, deletion or substitution; the modification of at least one base pair is sufficient.
According to an advantageous embodiment of the strain in accordance with the invention, the erp gene is modified by insertion of a nucleotide or polynucleotide which may be a selectable gene. This gene may in particular encode the resistance to an antibiotic such as kanamycin, spectinomycin or hygromycin.
The preferred Mycobacterium strains are those belonging to the Mycobacterium genus, preferably to the Mycobacterium tuberculosis complex and still more preferably to the Mycobacterium tuberculosis species or to the Mycobacterium bovis species.
The present invention relates more particularly to the BCG erp::Kn strain also called BCG erp::aph (Collection Nationale de Cultures de Microorganismes (xe2x80x9cCNCMxe2x80x9d), located at Institut Pasteur, 28, Rue du Docteur Roux, F-75724 PARIS CEDEX 15, France, Deposit No. I-1896, made Jul. 15, 1997) or a variant incapable of expressing the product of the active erp gene as well as the M. tuberculosis H37Rv erp::aph strain (CNCM Deposit No. I-2048, made Jun. 29, 1998) or a variant incapable of expressing the gene product.
The invention also relates to a Mycobacterium strain whose erp gene is modified and which is capable of producing, following recombination events, epitopes or antigenic determinants capable of immunizing and/or protecting against pathogenic agents such as infectious agents or cancer genes, or of producing molecules leading to a modulation of the immune system such as cytokines, chemokines, soluble receptors for molecules interacting with agents leading to a pathological condition or inducers of immune responses such as IL2, IL4, IL10 or IL12 (in humans or animals).
The present invention therefore also relates to a Mycobacterium strain as described above which is capable, in addition, of expressing a polynucleotide encoding a mycobacterium antigen of a species other than that to which said strain belongs, it being possible for the polynucleotide in question to be foreign to the Mycobacterium genus.
A subject of the invention is also a purified polynucleotide comprising a modified erp gene and a fragment of at least 60 nucleotides corresponding to the whole or part of a gene encoding an exported antigen of the Mycobacterium genus or encoding an antigen foreign to the Mycobacterium genus.
The modification of the erp gene may be obtained, for example, by addition, insertion or modification of nucleotides. In the context of the invention, the selection of the Mycobacterium strain whose erp gene is thus modified may be carried out by gene amplification and nucleotide sequencing or RFLP of the nucleic region mutated in said strain isolated on agar according to the counterselection protocol in the presence of sucrose (Pelicic et al., 1996), for example. An alternative consists in carrying out hybridizations under high stringency conditions (Berthet et al., 1995) characterized by the use of a probe corresponding to the whole or part of the erp gene which has been genetically modified but which conserved at least 20% of its activity and which preferably hybridizes with the whole or part of the modified gene present in the desired strain.
The modification of the erp gene may also be carried out by means of a recombinant vector comprising the inactivated erp gene. This vector is used for the transformation of a Mycobacterium strain and should allow an allelic exchange with the wild-type erp gene with the aim of modifying it.
Advantageously, the vector in accordance with the invention comprises a replication origin which is heat-sensitive in mycobacteria. It may also comprise the counterselectable sacb gene optionally with a gene allowing positive selection such as a gene encoding resistance to an antibiotic.
The modification of the erp gene in the vector in accordance with the invention may be carried out as described above.
More particularly, said vector corresponds to the recombinant plasmid pIPX56 (CNCM Deposit No. I-1895, made Jul. 15, 1997). Indeed, this plasmid consists of an E. colixe2x80x94mycobacteria shuttle cloning vector of the pPR27 type (deposited at CNCM under the number I-1730) comprising a replication origin which is heat-sensitive in mycobacteria, the counterselectable sacB gene and conferring resistance to gentamycin. In the plasmid plPX56, an insertion of 5.1 kb of a PstI fragment was carried out at the level of the unique PstI site of pPR27. This 5.1 kb fragment corresponds to a 3.9 kb DNA fragment of M. tuberculosis comprising the erp gene into which a cassette (1.2 kb) conferring resistance to kanamycin has been inserted. This plasmid therefore makes it possible to carry out allelic exchange experiments at the level of the erp locus in mycobacteria.
In the context of the present invention, it is also advantageous to be able to have a vector derived from pIPX56 comprising the unmodified erp gene.
The subject of the invention is therefore also the use of a recombinant vector as described above for the preparation of a Mycobacterium strain in accordance with the invention by allelic exchange.
Another subject of the invention is a method for the production of a Mycobacterium strain as described above comprising the steps of:
transforming, with a vector as described above, a Mycobacterium strain propagated at a permissive temperature,
culturing the colonies resulting from the transformation on a medium supplemented with a selectable product and sucrose,
isolating the recombinant strain.
According to an advantageous embodiment of the method in accordance with the invention, the selectable product is an antibiotic such as kanamycin, spectinomycin or hygromycin.
By way of example, a recombinant Mycobacterium strain is produced in accordance with the invention as follows:
a) the plasmid pIPX56 is introduced by electroporation into a strain of the Mycobacterium tuberculosis complex propagated at a permissive temperature (32xc2x0 C.). This step makes it possible to have a population of bacteria in which each individual possesses several copies of the erp::Kn cassette;
b) a colony resulting from the transformation is cultured in liquid medium at 32xc2x0 C. for 10 days, and then the culture is inoculated on plates containing kanamycin (50 mg/ml) and 2% sucrose (weight/vol.) which are incubated at a nonpermissive temperature at 39xc2x0 C. This step makes it possible to enrich in double homologous recombination events by counterselection and elimination of the integrations of vectors (single homologous recombination or illegitimate recombination).
The invention also relates to an immunogenic composition comprising a Mycobacterium strain in accordance with the invention or obtained by carrying out the method mentioned above.
It also relates to a vaccine composition comprising a Mycobacterium strain in accordance with the invention or obtained by carrying out the method mentioned above, in combination with at least one pharmaceutically compatible excipient.
This vaccine composition is intended for the immunization of humans and animals against a pathogenic strain of mycobacteria and comprises an immunogenic composition as described above in combination with a pharmaceutically compatible excipient (such as a saline buffer), optionally in combination with at least one immunity adjuvant such as aluminum hydroxide or a compound belonging to the muramyl peptide family.
To obtain an adjuvant effect for the vaccine, many methods envisage the use of agents such as aluminum hydroxide or phosphate (alum) which are commonly used as a solution titrating 0.05 to 0.01% in a phosphate-buffered saline, mixed with synthetic polymers of sugar (Carbopol) as a 0.25% solution. Another suitable adjuvant compound is DDA (2 dimethyldioctadecylammonium bromide), as well as immunomodulatory substances such as the lymphokines (for example gamma-IFN, IL-1, IL-2 and IL-12) or also gamma-IFN-inducing compounds such as poly I:C.
The vaccine composition in accordance with the present invention is advantageously prepared in injectable form, for administration orally or by inhalation, or in liquid solution or in suspension; suitable solid forms intended to be prepared in solution or in liquid suspension before injection can also be prepared.
Furthermore, the vaccine composition may contain minor components of an auxiliary substance such as wetting or emulsifying agents, agents for buffering the pH or adjuvants which stimulate the efficacy of the vaccines.
The vaccine compositions of the invention are administered in a manner compatible with the dosage formulation and in a therapeutically effective and immunogenic quantity. The quantity to be administered depends on the subject to be treated, including, for example, the individual capacity of their immune system to induce an immune response.
The vaccine dosage will depend on the route of administration and will vary according to the age of the patient to be vaccinated and, to a lesser degree, the size of this patient. Preferably, the vaccine composition according to the present invention is administered by the intradermal route in a single portion or by the oral route or by aerosol.
In some cases, it will be necessary to carry out multiple administrations of the vaccine composition in accordance with the present invention without, however, generally exceeding six administrations, preferably four vaccinations. The successive administrations will normally be made at an interval of 2 to 12 weeks, preferably of 3 to 5 weeks. Periodic boosters at intervals of 1 to 5 years, preferably 3 years, are desirable in order to maintain the desired level of protective immunity.
The invention also relates to a diagnostic method which makes it possible to discriminate between individuals, on the one hand, who have been vaccinated with the aid of a Mycobacterium strain no longer producing active ERP and, on the other hand, those who have had a natural infection or a vaccination with the aid of a strain producing the natural ERP protein.
Indeed, the individuals who have had a vaccination with a Mycobacterium strain no longer producing the natural ERP protein can be distinguished by the absence, from a biological sample such as for example serum, of antibodies directed against ERP and/or by the absence of T reactivity (measured for example during a test of proliferation or a test of secretion of cytokines or CTL test) against the purified ERP protein. An alternative also consists in testing for a differential reactivity with the aid of antibodies directed against the unmodified part of the natural ERP protein compared with the corresponding part of the mutated ERP protein.
The subject of the present invention is therefore also a method of screening individuals, to whom a vaccine composition in accordance with the invention has been administered, comprising detecting the absence, from a biological sample from said individuals, of antibodies or of T cells directed against the whole or part of the purified ERP protein, it being possible for the biological sample to be blood.
The subject of the invention is also a composition comprising the modified ERP protein.
Another aspect of the present invention relates to the repeat sequences present in the erp gene in particular of the strains of the M. tuberculosis complex. Indeed, in the majority of the cases studied by the Inventors, the tuberculosis patients did not develop a humoral response against erp. The mice vaccinated with BCG do not develop a humoral response against erp either. By contrast, the leprosy patients develop a strong response against erp. The major difference between the ERP protein from M. tuberculosis and the similar protein from M. leprae lies in the absence of repeats in M. leprae. 
Consequently, the repeats may be responsible for the blocking of the humoral response specifically against erp or even, in general, against other antigens. It is indeed known that tuberculosis patients develop only a weak humoral response at the beginning of the tuberculosis disease. It could therefore be possible to use the repeats carried by erp to inhibit the development of a specific humoral response by combining these repeats with any antigen against which it is desired to avoid a humoral response being induced or perhaps even used by these repeats to inhibit any humoral response in some advantageous contexts. This type of strategy could be appropriate for the following situations: avoiding the development of the humoral response against viral vaccine vectors (see table).
Thus, the present invention relates to the use of the repeat sequences of the erp gene, optionally in combination with at least one other antigen, for inhibiting the development of a humoral response.
It also relates to a vector for expression in a microorganism, characterized in that it comprises a nucleotide sequence encoding the ERP protein lacking its repeat sequences. The microorganism harboring the expression vector may be, for example, E. coli or any other organism which may be suitable for the expression of a nucleotide sequence encoding the ERP protein lacking its repeat sequences, including the mycobacteria.
The subject of the present invention is also a strain of mycobacteria, characterized in that the erp gene lacks its repeat sequences. Indeed, such a strain, having ERP with no repeats, would be immunogenic while having a protective effect.